Tissue volume reduction

ABSTRACT

Devices, compositions, and methods for achieving non-surgical lung volume reduction (e.g., bronchoscopic lung volume reduction (BLVR)) are described. BLVR can be carried out by collapsing a region of the lung, adhering one portion of the collapsed region to another, and promoting fibrosis in or around the adherent tissue.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation application of and claimspriority to U.S. application Ser. No. 09/379,460, filed on Aug. 23,1999, now U.S. Pat. No. 6,610,043, which is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

[0002] The field of the invention is tissue volume reduction, forexample, lung volume reduction.

SUMMARY OF THE INVENTION

[0003] End stage emphysema can be treated with lung volume reductionsurgery (LVRS) (see, e.g., Cooper et al., J. Thorac. Cardiovasc. Surg.109:106-116, 1995). While it may seem counter-intuitive that respiratoryfunction would be improved by removing part of the lung, excisingover-distended tissue (as seen in patients with heterogeneous emphysema)allows adjacent regions of the lung that are more normal to expand. Inturn, this expansion allows for improved recoil and gas exchange. Evenpatients with homogeneous emphysema benefit from LVRS because resectionof abnormal lung results in overall reduction in lung volumes, anincrease in elastic recoil pressures, and a shift in the staticcompliance curve towards normal (Hoppin, Am. J. Resp. Crit. Care Med.155:520-525, 1997).

[0004] While many patients who have undergone LVRS experiencesignificant improvement (Cooper et al., J. Thorac. Cardiovasc. Surg.112:1319-1329, 1996), they have assumed substantial risk. LVRS iscarried out by surgically removing a portion of the diseased lung, whichhas been accessed either by inserting a thoracoscope through the chestwall or by a more radical incision along the sternum (Katloff et al.,Chest 110:1399-1406, 1996). Thus, gaining access to the lung istraumatic, and the subsequent procedures, which can include stapling thefragile lung tissue, can cause serious post-operative complications.

SUMMARY OF THE INVENTION

[0005] The invention features devices, compositions, and methods forachieving non-surgical lung volume reduction. In one aspect, the methodsare carried out using a bronchoscope, which completely eliminates theneed for surgery because it allows the tissue reduction procedure to beperformed through the patient's trachea and smaller airways. In thisapproach, bronchoscopic lung volume reduction (BLVR) is performed bycollapsing a region of the lung, adhering one portion of the collapsedregion to another, and promoting fibrosis in or around the adherenttissue.

[0006] Preferred embodiments may include one or more of the followingfeatures.

[0007] There are numerous ways to induce lung collapse. For example, amaterial that increases the surface tension of fluids lining the alveoli(i.e., a material that can act as an anti-surfactant) can be introducedthrough the bronchoscope (preferably, through a catheter lying withinthe bronchoscope). The material can include fibrinogen, fibrin, orbiologically active fragments thereof. Lung collapse can also be inducedby blocking air flow into and out of the region of the lung that istargeted for collapse. This is achieved by inserting a balloon catheterthrough the bronchoscope and inflating the balloon so that it occludesthe bronchus or bronchiole into which it has been placed.

[0008] Similarly, there are numerous ways to promote adhesion betweenone portion of the collapsed lung and another. If fibrinogen is selectedas the anti-surfactant, adhesion is promoted by exposing the fibrinogento a fibrinogen activator, such as thrombin, which cleaves fibrinogenand polymerizes the resulting fibrin. Other substances, includingthrombin receptor agonists and batroxobin, can also be used to activatefibrinogen. If fibrin is selected as the anti-surfactant, no additionalsubstance or compound need be administered; fibrin can polymerizespontaneously, thereby adhering one portion of the collapsed tissue toanother.

[0009] Fibrosis is promoted by providing one or more polypeptide growthfactors together with one or more of the anti-surfactant or activatorsubstances described above. The growth factors can be selected from thefibroblast growth factor (FGF) family or can be transforming growthfactor beta-like (TGF-β-like) polypeptides.

[0010] The compositions described above can also contain one or moreantibiotics to help prevent infection. Alternatively or in addition,antibiotics can be administered via other routes (e.g., they may beadministered orally or intramuscularly).

[0011] Other aspects of the invention include the compositions describedabove for promoting collapse and/or adhesion, as well as devices forintroducing the composition into the body. For example, in one aspect,the invention features physiologically acceptable compositions thatinclude a polypeptide growth factor or a biologically active fragmentthereof (e.g., a platelet-derived growth factor, a fibroblast growthfactor (FGF), or a transforming growth factor-β-like polypeptide) andfibrinogen, or a fibrin monomer (e.g., a fibrin I monomer, a fibrin IImonomer, a des BB fibrin monomer, or any mixture or combinationthereof), or a fibrinogen activator (e.g., thrombin). The fibrinogen,fibrin monomers, and fibrinogen activators useful in BLVR can bebiologically active mutants (e.g., fragments) of these polypeptides.

[0012] In another aspect, the invention features devices for performingnon-surgical lung volume reduction. For example, the invention featuresa device that includes a bronchoscope having a working channel and acatheter that can be inserted into the working channel. The catheter cancontain multiple lumens and can include an inflatable balloon. Anotherdevice for performing lung volume reduction includes a catheter having aplurality of lumens (e.g., two or more) and a container for materialhaving a plurality of chambers (e.g., two or more), the chambers of thecontainer being connectable to the lumens of the catheter. These devicescan also include an injector to facilitate movement of material from thecontainer to the catheter.

[0013] BLVR has several advantages over standard surgical lung volumereduction (LVRS). BLVR should reduce the morbidity and mortality knownto be associated with LVRS (Swanson et al., J. Am. Coll. Surg.185:25-32, 1997). Atrial arrhythmias and prolonged air leaks, which arethe most commonly reported complications of LVRS, are less likely tooccur with BLVR because BLVR does not require stapling of fragile lungtissue or surgical manipulations that irritate the pericardium. BLVR mayalso be considerably less expensive than SLVR, which currently costsbetween approximately $18,000 and $26,000 per case. The savings would betremendous given that emphysema afflicts between two and six millionpatients in America alone. In addition, some patients who would not becandidates for LVRS (due, e.g., to their advanced age) may undergo BLVR.Moreover, should the need arise, BLVR affords patients an opportunity toundergo more than one volume reduction procedure. While repeat surgicalintervention is not a viable option for most patients (because ofpleural adhesions that form following the original procedure), no suchlimitation should exist for patients who have undergone BLVR.

[0014] Other features and advantages of the invention will be apparentfrom the following detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 is a schematic representation of BLVR.

[0016]FIG. 2A illustrates a catheter that can be inserted through abronchoscope.

[0017]FIG. 2B is a cross-sectional view through the shaft of thecatheter illustrated in FIG. 2A.

[0018]FIG. 2C illustrates a cartridge that can be attached to thecatheter illustrated in FIG. 2A.

[0019]FIG. 2D illustrates an injector that can be used to expel materialfrom the cartridge illustrated in FIG. 2C.

[0020]FIG. 2E illustrates the catheter of FIG. 2A assembled with thecartridge of FIG. 2C, the injector of FIG. 2D, and a leur-lock,air-filled syringe.

[0021]FIGS. 3A and 3B are graphs depicting the surface tension vs.surface area of surfactant films from a control guinea pig (FIG. 3A) anda guinea pig exposed to LPS (FIG. 3B).

[0022]FIGS. 4A and 4B are bar graphs depicting the surface filmstability parameter (Gy/dA)l(A/y) as a function of protein/lipidconcentration for fibrinogen and albumin surfactant mixtures.

[0023]FIGS. 5A and 5B are bar graphs depicting dynamic (FIG. 5A) andquasi-static (FIG. 5B) compliance 3 months after sheep were exposed toPapain. n=6.

[0024]FIGS. 6A and 6B are graphs plotting the relationship betweenphysiology (Cdyn, as a % baseline, is shown in FIG. 6A and R_(L), alsoas a % baseline, is shown in FIG. 6B) and emphysema severity score.

[0025]FIG. 7 is a graph illustrating static lung compliance (volume inliters vs. Ptp in cm H₂O) at baseline (i.e., pre-treatment) and at eightweeks following papain therapy in sheep.

DETAILED DESCRIPTION

[0026] Lung volume can be reduced non-surgically using the devices,compositions, and methods described herein. For example, lung volume canbe reduced using a bronchoscope (bronchoscopic lung volume reduction isabbreviated herein as BLVR). Referring to FIG. 1, a flexiblebronchoscope 10 is inserted through a patient's trachea 12 to a targetregion 20 a of the lung 20, and a balloon catheter 50 with a distallumen port 60 (FIG. 2) is inserted through a channel within thebronchoscope. Target region 20 a will collapse either when the airpassage 14 to target region 20 a is occluded or when an anti-surfactantis administered through balloon catheter 50 to target region 20 a.Regardless of the cause of collapse, one portion of the collapsed targetregion will adhere to another when exposed to one or more of thecompositions described below. These compositions include substances thatcan polymerize either spontaneously (e.g., fibrin) or in response to anactivator (e.g., fibrinogen). In addition, one or more of thecompositions contains a polypeptide growth factor that promotesfibrosis, and may contain an antibiotic to help prevent infection or anadditional factor (such as factor XIIIa transglutaminase) to promotepolymerization. Following application of the composition(s), thebronchoscope is removed.

[0027] Patients who have chronic obstructive pulmonary disease canbenefit from BLVR. These patients include, but are not limited to, thosewho have emphysema, chronic asthma, chronic bronchitis, andbrochiectasis. BLVR can also be performed when a patient's lung isdamaged by trauma or in the event of a spontaneous pneumothorax.

[0028] Identifying and Gaining Access to a Target Region of the Lung

[0029] Once a patient is determined to be a candidate for BLVR, thetarget region 20 a of the lung that will be removed can be identifiedusing radiological studies (e.g., chest X-rays) and computed tomographyscans. When the procedure is subsequently performed, the patient isanesthetized and intubated, and can be placed on an absorbable gas (e.g.at least 90% oxygen and up to 100% oxygen) for a specified period oftime (e.g., approximately 30 minutes). The region(s) of the lung thatwere first identified radiologically are then identifiedbronchoscopically.

[0030] Suitable bronchoscopes include those manufactured by Pentax,Olympus, and Fujinon, which allow for visualization of an illuminatedfield. The physician guides bronchoscope 10 into trachea 12 and throughthe bronchial tree so that the open tip 60 of bronchoscope 10 ispositioned at the entrance to target region 20 a (i.e., to the region ofthe lung that will be reduced in volume). Bronchoscope 10 can be guidedthrough progressively narrower branches of the bronchial tree to reachvarious subsegments of either lung 20. For example, as shown in FIG. 1,the bronchoscope can be guided to a subsegment within the upper lobe ofthe patient's left lung.

[0031] The balloon catheter 50 mentioned above (and described more fullybelow) is then guided through bronchoscope 10 to target region 20 a oflung 20. When catheter 50 is positioned within bronchoscope 10, balloon58 is inflated so that material passed through the catheter will becontained in regions of the lung distal to the balloon. The targetedregion can be lavaged with saline to reduce the amount of surfactantthat is naturally present, and a physiologically compatible compositioncontaining an anti-surfactant (i.e., an agent that increases the surfacetension of fluids lining the alveoli) is applied to the targeted regionof the lung through the catheter. Preferably, the composition isformulated as a solution or suspension and includes fibrin orfibrinogen. An advantage of administering these substances is that theycan each act not only as anti-surfactants, but can participate in theadhesive process as well.

[0032] Fibrinogen-Based Solutions

[0033] Fibrinogen can function as an anti-surfactant because itincreases the surface tension of fluids lining the alveoli, and it canfunction as a sealant or adhesive because it can participate in acoagulation cascade in which it is converted to a fibrin monomer that isthen polymerized and cross-linked to form a stable mesh. Fibrinogen,which has also been called Factor I, represents about 2-4 g/L of bloodplasma protein, and is a monomer that consists of three pairs ofdisulfide-linked polypeptide chains designated (Aα)₂, (Bβ)₂, and γ₂. The“A” and “B” chains represent the two small N-terminal peptides and arealso known as fibrinopeptides A and B, respectively. The cleavage offibrinogen by thrombin results in a compound termed fibrin I, and thesubsequent cleavage of fibrinopeptide B results in fibrin II. Althoughthese cleavages reduce the molecular weight of fibrinogen only slightly,they nevertheless expose the polymerization sites. In the process ofnormal clot formation, the cascade is initiated when fibrinogen isexposed to thrombin, and this process can be replicated in the contextof lung volume reduction when fibrinogen is exposed to an activator suchas thrombin, or an agonist of the thrombin receptor, in an aqueoussolution containing calcium (e.g. 1.5 to 5.0 mM calcium).

[0034] The fibrinogen-containing composition can include 3-12%fibrinogen and, preferably, includes approximately 10% fibrinogen insaline (e.g., 0.9% saline) or another physiologically acceptable aqueoussolution. The volume of anti-surfactant administered will vary,depending on the size of the region of the lung, as estimated fromreview of computed tomagraphy scanning of the chest. For example, thetargeted region can be ravaged with 10-100 mls (e.g., 50 mls) offibrinogen solution (10 mg/ml). To facilitate lung collapse, the targetregion can be exposed to (e.g., rinsed or lavaged with) an unpolymerizedsolution of fibrinogen and then exposed to a second fibrinogen solutionthat is subsequently polymerized with a fibrinogen activator (e.g.,thrombin or a thrombin receptor agonist).

[0035] The anti-surfactant can contain fibrinogen that was obtained fromthe patient before the non-surgical lung reduction procedure commenced(i.e., the anti-surfactant or adhesive composition can includeautologous fibrinogen). The use of an autologous substance is preferablebecause it eliminates the risk that the patient will contract some formof hepatitis (e.g., hepatitis B or non A, non B hepatitis), an acquiredimmune deficiency syndrome (AIDS), or other blood-transmitted infection.These infections are much more likely to be contracted when thefibrinogen component is extracted from pooled human plasma (see, e.g.,Silberstein et al., Transfusion 28:319-321, 1988). Human fibrinogen iscommercially available through suppliers known to those of skill in theart or may be obtained from blood banks or similar depositories.

[0036] Polymerization of fibrinogen-based anti-surfactants can beachieved by adding a fibrinogen activator. These activators are known inthe art and include thrombin, batroxobin (such as that from B. Moojeni,B. Maranhao, B. atrox, B. Ancrod, or A. rhodostoma), and thrombinreceptor agonists. When combined, fibrinogen and fibrinogen activatorsreact in a manner similar to the final stages of the natural bloodclotting process to form a fibrin matrix. More specifically,polymerization can be achieved by addition of thrombin (e.g., 1-10 unitsof thrombin per ng of fibrinogen). If desired, 1-5% (e.g., 3%) factorXIIIa transglutaminase can be added to promote cross-linking.

[0037] In addition, to promote fibrosis (or scarring) at the site whereone region of the collapsed lung adheres to another, one or more of thecompositions applied to achieve lung volume reduction (e.g., thecomposition containing fibrinogen) can contain a polypeptide growthfactor. Numerous factors can be included. Platelet-derived growth factor(PDGF) and those in the fibroblast growth factor and transforming growthfactor-β families are preferred.

[0038] For example, the polypeptide growth factor included in acomposition administered to reduce lung volume (e.g., the fibrinogen-,fibrinogen activator-, or fibrin-based compositions described herein)can be basic FGF (bFGF), acidic FGF (aFGF), the hst/Kfgf gene product,FGF-5, FGF-10, or int-2. The nomenclature in the field of polypeptidegrowth factors is complex, primarily because many factors have beenisolated independently by different researchers and, historically, namedfor the tissue type used as an assay during purification of the factor.This complexity is illustrated by basic FGF, which has been referred toby at least 23 different names (including leukemic growth factor,macrophage growth factor, embryonic kidney-derived angiogenesis factor2, prostatic growth factor, astroglial growth factor 2, endothelialgrowth factor, tumor angiogenesis factor, hepatoma growth factor,chondrosarcoma growth factor, cartilage-derived growth factor 1,eye-derived growth factor 1, heparin-binding growth factors class II,myogenic growth factor, human placenta purified factor, uterine-derivedgrowth factor, embryonic carcinoma-derived growth factor, humanpituitary growth factor, pituitary-derived chondrocyte growth factor,adipocyte growth factor, prostatic osteoblastic factor, and mammarytumor-derived growth factor). Thus, any factor referred to by one of theaforementioned names is within the scope of the invention.

[0039] The compositions can also include “functional polypeptide growthfactors,” i.e., growth factors that, despite the presence of a mutation(be it a substitution, deletion, or addition of amino acid residues)retain the ability to promote fibrosis in the context of lung volumereduction. Accordingly, alternate molecular forms of polypeptide growthfactors (such as the forms of bFGF having molecular weights of 17.8,22.5, 23.1, and 24.2 kDa) are within the scope of the invention (thehigher molecular weight forms being colinear N-terminal extensions ofthe 17.8 kDa bFGF (Florkiewicz et al., Proc. Natl. Acad. Sci. USA86:3978-3981, 1989)).

[0040] It is well within the abilities of one of ordinary skill in theart to determine whether a polypeptide growth factor, regardless ofmutations that affect its amino acid content or size, substantiallyretains the ability to promote fibrosis as would the full length, wildtype polypeptide growth factor (i.e., whether a mutant polypeptidepromotes fibrosis at least 40%, preferably at least 50%, more preferablyat least 70%, and most preferably at least 90% as effectively as thecorresponding wild type growth factor). For example, one could examinecollagen deposition in cultured fibroblasts following exposure tofull-length growth factors and mutant growth factors. A mutant growthfactor substantially retains the ability to promote fibrosis when itpromotes at least 40%, preferably at least 50%, more preferably at least70%, and most preferably at least 90% as much collagen deposition asdoes the corresponding, wild-type factor. The amount of collagendeposition can be measured in numerous ways. For example, collagenexpression can be determined by an immunoassay. Alternatively, collagenexpression can be determined by extracting collagen from fibroblasts(e.g., cultured fibroblasts or those in the vicinity of the reduced lungtissue) and measuring hydroxyproline.

[0041] The polypeptide growth factors useful in the invention can benaturally occurring, synthetic, or recombinant molecules and can consistof a hybrid or chimeric polypeptide with one portion, for example, beingbFGF or TGFβ, and a second portion being a distinct polypeptide. Thesefactors can be purified from a biological sample, chemicallysynthesized, or produced recombinantly by standard techniques (see,e.g., Ausubel et al., Current Protocols in Molecular Biology, New York,John Wiley and Sons, 1993; Pouwels et al., Cloning Vectors: A LaboratoryManual, 1985, Supp. 1987).

[0042] Of course, various fibrosis-promoting growth factors can be usedin combination.

[0043] One of ordinary skill in the art is well able to determine thedosage of a polypeptide growth factor required to promote fibrosis inthe context of BLVR. The dosage required can vary and can range from1-100 nM.

[0044] In addition, any of the compositions or solutions describedherein for lung volume reduction (e.g., the fibrinogen-based compositiondescribed above) can contain one or more antibiotics (e.g., ampicillin,gentamycin, cefotaxim, nebacetin, penicillin, or sisomicin). Theinclusion of antibiotics in therapeutically applied compositions is wellknown to those of ordinary skill in the art.

[0045] Fibrin-Based Solutions

[0046] Fibrin can also function as an anti-surfactant as well as asealant or adhesive. However, in contrast to fibrinogen, fibrin can beconverted to a polymer without the application of an activator (such asthrombin or factor XIIIa). In fact, fibrin I monomers can spontaneouslyform a fibrin I polymer that acts as a clot, regardless of whether theyare crosslinked and regardless of whether fibrin I is further convertedto fibrin II polymer. Without expressing an intention to limit theinvention to compounds that function by any particular mechanism, it canbe noted that when fibrin I monomers come into contact with a patient'sblood, the patient's own thrombin and factor XIII may convert the fibrinI polymer to crosslinked fibrin II polymer.

[0047] Any form of fibrin monomer that can be converted to a fibrinpolymer can be formulated as a solution and used for lung volumereduction. For example, fibrin-based compositions can contain fibrin Imonomers, fibrin II monomers, des BB fibrin monomers, or any mixture orcombination thereof. Preferably, the fibrin monomers are notcrosslinked.

[0048] Fibrin can be obtained from any source so long as it is obtainedin a form that can be converted to a fibrin polymer (similarly,non-crosslinked fibrin can be obtained from any source so long as it canbe converted to crosslinked fibrin). For example, fibrin can be obtainedfrom the blood of a mammal, such as a human, and is preferably obtainedfrom the patient to whom it will later be administered (i.e., the fibrinis autologous fibrin). Alternatively, fibrin can be obtained from cellsthat, in culture, secrete fibrinogen.

[0049] Fibrin-based compositions can be prepared as described in U.S.Pat. No. 5,739,288 (which is hereby incorporated by referenced in itsentirety), and can contain fibrin monomers having a concentration of noless than about 10 mg/ml. For example, the fibrin monomers can bepresent at concentrations of from about 20 mg/ml to about 200 mg/ml;from about 20 mg/ml to about 100 mg/ml; and from about 25 mg/ml to about50 mg/ml.

[0050] The spontaneous conversion of a fibrin monomer to a fibrinpolymer may be facilitated by contacting the fibrin monomer with calciumions (as found, e.g., in calcium chloride, e.g., a 3-30 mM CaCl₂solution). Except for the first two steps in the intrinsic bloodclotting pathway, calcium ions are required to promote the conversion ofone coagulation factor to another. Thus, blood will not clot in theabsence of calcium ions (but, in a living body, calcium ionconcentrations never fall low enough to significantly affect thekinetics of blood clotting; a person would die of muscle tetany beforecalcium is diminished to that level). Calcium-containing solutions(e.g., sterile 10% CaCl₂) can be readily made or purchased from acommercial supplier.

[0051] The fibrin-based compositions described here can also include oneor more polypeptide growth factors that promote fibrosis (or scarring)at the site where one region of the collapsed lung adheres to another.Numerous factors can be included and those in the fibroblast growthfactor and transforming growth factor-β families are preferred. Thepolypeptide growth factors suitable for inclusion with fibrin-basedcompositions include all of those (described above) that are suitablefor inclusion with fibrinogen-based compositions.

[0052] Application of Fibrin- and Fibrinogen-Based CompositionsFollowing Lung Collapse

[0053] While a targeted region of the lung can be collapsed by exposureto one of the substances described above, these substances can also beapplied to adhere one region of the lung to another (and to promotefibrosis) when the collapse has been induced by other means. Forexample, the substances described above can be applied after the lungcollapses from blockage of airflow into or out of the targeted region.Such blockage can be readily induced by, for example, inserting abronchoscope into the trachea of an anesthetized patient, inserting aballoon catheter through the bronchoscope, and inflating the balloon sothat little or no air passes into the targeted region of the lung.Collapse of the occluded region after the lung is filled with absorbablegas would occur over approximately 5-15 minutes, depending on the sizeof the region occluded. Alternatively, the target region could beAlternatively, a fibrinogen- or fibrin-based solution can be appliedafter the lung is exposed to another type of anti-surfactant (e.g., anon-toxic detergent).

[0054] A Catheter for Application of Material to the Lung

[0055] Referring to FIG. 2A, any of the substances described above canbe administered to the lung by a balloon catheter 50 having multipleports 52 through which materials (such as solutions or suspensions) orgases (such as air) can be injected via a corresponding number oflumens. The ports of catheter 50 are arranged as follows. A first port54 having a proximal end 54 a adapted for connection with a gas supply(e.g., a leur-lock syringe containing air) communicates with internallumen 56 of catheter 50, which terminates within inflatable balloon 58near distal tip 60 of catheter 50. A second port 64 having a proximalend 64 a adapted for connection with a source of one or more materials(e.g., medication cartridge 80, described below) communicates withinternal lumen 66, which terminates at open distal tip 60 of catheter50. A third port 74 having a proximal end 74 a adapted for connectionwith a source of one or more materials (e.g., medication cartridge 80)communicates with internal lumen 76, which also terminates at opendistal tip 60 of catheter 50.

[0056] Thus, gas injected through port 54 travels through internal lumen56 to inflate balloon 58, and material injected through port 64 and/orport 74 travels through internal lumens 66 and 76, respectively, todistal tip 60 of catheter 50. Upon reaching distal tip 60 of catheter50, materials previously separated within lumens 66 and 76 would mixtogether.

[0057] Referring to FIG. 2B, internal lumen 54, internal lumen 64, andinternal lumen 74 are shown in a cross-sectional view of shaft 51 ofcatheter 50. In another embodiment, lumens 66 and 76 can differ in size,with the diameter of the lumen through which the fibrinogen-basedsolution is applied being approximately twice as great as the diameterof the lumen through which the solution containing the fibrinogenactivator is applied.

[0058] Referring to FIG. 2C, cartridge 80 can be attached to catheter 50to inject material via ports 64, 74 and lumens 66, 76. Cartridge 80includes a first chamber 82 and a second chamber 92, either or both ofwhich can contain material useful in BLVR (e.g., chamber 82 can containa mixture of fibrinogen, TGF-β, and gentamycin, and chamber 92 cancontain thrombin in a calcium-buffered solution). Material withincartridge 80 can be administered to the lung by way of catheter 20, asfollows. Upper wall 84 of chamber 82 includes orifice 84 a, throughwhich pressure can be applied to depress plunger 86. Depression ofplunger 86 forces material within chamber 82 toward lower wall 88 ofchamber 82, through opening 88 a, and, when cartridge 80 is attached tocatheter 50, into port 64 of catheter 50. Similarly, upper wall 94 ofchamber 92 includes orifice 94 a, through which pressure can be appliedto depress plunger 96. Depression of plunger 96 forces material withinchamber 92 toward lower wall 98 of chamber 92, through opening 98 a,and, when cartridge 80 is attached to catheter 50, into port 74 ofcatheter 50.

[0059] Referring to FIG. 2D, to aid the transfer of material fromcartridge 80 to catheter 50, cartridge 80 can be placed within recess 45of a frame-shaped injector 40. Injector 40 includes upper wall 42,having orifices 42 a and 42 b, through which arm 44 is inserted. Prong44 a of arm 44 enters injector 40 through orifice 42 a and prong 44 b ofarm 44 enters injector 40 through orifice 42 b. When cartridge 80 isplaced within injector 40 and arm 44 is depressed, prongs 44 a and 44 bare forced against plungers 86 and 96, respectively, thereby extrudingmaterials in chambers 82 and 92 through openings 88 a and 98 a,respectively, of cartridge 80 and openings 46 a and 46 b, respectively,of lower wall 46 of injector 40.

[0060]FIG. 2E illustrates catheter 50 assembled with cartridge 80,injector 40, and a leur-lock, air-filled syringe 30.

[0061] The preferred methods, materials, and examples that will now bedescribed are illustrative only and are not intended to be limiting;materials and methods similar or equivalent to those described hereincan be used in the practice or testing of the invention.

EXAMPLE 1 BLVR in an Isolated Calf Lung

[0062] Isolated calf lungs are excellent models for BLVR because theyare easy to work with and anatomically similar to human lungs. Calflungs having 4-5 liters total lung capacity were purchased from Arenaand Sons' Slaughter House (Hopkinton, Mass.) and delivered on ice to thelaboratory within 3 hours of procurement. The lungs were tracheallycannulated with a #22 tubing connector and suspended from a ring clampwith the diaphragmatic surface resting in a large Teflon dish containing2-3 mm of phosphate buffered saline (PBS). The visceral pleural surfacewas kept moist by spraying it with a mist of 0.15 M NaCl at regularintervals. Pleural leaks were identified by the appearance of bubbles onthe pleural surface and by assessing the lungs' ability to hold aconstant pressure of 20 cm H₂O inflation pressure. Leaks were sealed byautologous buttress plication. Any adversely affected sections of thelungs were rolled up and stapled in a manner similar to that used inLVRS in humans (Swanson et al., J. Am. Coll. Surg. 185:25-32, 1997).

[0063] Absolute lung volumes were measured by gas dilution usingnitrogen as the tracer gas (Conrad et al., Pulmonary FunctionTesting—Principles and Practice, Churchill Livingstone Publishers, NewYork, N.Y., 1984). Measurements were performed at 0 cm H₂Otranspulmonary pressure as follows. A three liter syringe was filledwith 1.5 liters of 100% oxygen from a reservoir bag. The isolated lung,containing an unknown volume of room air (79% nitrogen) was thenconnected in-line with the syringe containing 0% nitrogen via a threeway valve. The gas was mixed well by depressing the plunger of thesyringe 60-100 times, and the equilibrium concentration of nitrogen wasthen determined using a nitrogen meter (Medtronics, Model 830 Nitrogenmeter). The unknown starting lung volume of room air was then calculatedaccording to the following conservation of mass equation:

VL={F _(N2f)/(0.79−FN2 f}·1.5 L

[0064] where F_(N2f) is the fraction of nitrogen measured at steadystate following mixing with 1.5 liters of oxygen from the syringe. Thismeasurement defines the single absolute lung volume that is required tocharacterize static lung mechanics.

[0065] Quasi-static deflation pressure volume curves (QSPVC) were thenrecorded during step-wise deflation from 20 cm H₂O to 0 cm H₂Otranspulmonary pressure as follows. Lungs were filled with air to 20 cmH₂O transpulmonary pressure, and the trachea was then occluded manually.Transpulmonary pressure was recorded using a 50 cm H₂O pressuretransducer positioned at the airway opening. Expired lung volume wasmeasured using a pneumotachograph (Hans Rudolf Inc, Kansas City, Mo.)connected in series with the tracheal cannula. Pressure as a function ofexpired lung volume (referenced to the starting volume at 20 cm H₂O) wasdetermined by intermittently occluding the trachea. Occlusions weremaintained long enough to allow for equilibration of tracheal andalveolar pressures (no change in tracheal pressure over three seconds).By combining the single absolute lung volume measurement made bynitrogen dilution at zero transpulmonary pressure with QSPVC data,complete static recoil pressure volume relationships were determined.These relationships can be described as an exponential functionaccording to the equation of Salazar et al. (J. Appl. Physiol.19:97-104, 1964):

V(P)=V _(max) −Ae ^(−kP)

[0066] where V is lung volume as a function of transpulmonary pressure;P is transpulmonary pressure; V_(max) is the extrapolated lung volume atinfinite pressure (approximately equal to TLC); A is the differencebetween V_(max) and the volume of gas trapped within the lung at zerotranspulmonary pressure (approximately equal to vital capacity); and kis the shape factor which describes the curvature of the exponentialrelationship between pressure and volume independent of the absolutevolume of the lung. The parameters V_(max), A, and k were determinedfrom a best fit linear regression analysis, and recoil pressure at totallung capacity (PTLC) determined by direct measurement.

[0067] It is useful to express the pressure volume relationship in termsof the parameters described above because each parameter is known tochange in a characteristic fashion in emphysema. Thus, one cananticipate specific changes following interventions designed to eitherproduce emphysema (e.g. papain exposure in the animal model) or correctthe abnormalities of emphysema (e.g., volume reduction; see Gibson etal., Am. Rev. Resp. Dis. 120:799-811, 1979). For example, V_(max)increases in emphysema due to lung hyper-expansion (this reflects anincrease in total lung capacity); k, the shape factor, also increasesdue to a decrease in the slope of the pressure volume relationship atlow lung volumes; and A, the difference between maximal lung volume andtrapped lung gas at zero transpulmonary pressure, decreases becausetrapped gas increases out of proportion to total lung capacity. Theseabnormalities will improve following effective lung volume reduction.

[0068] Following completion of lung volume and QSPVC measurements, lungfunction was assessed during simulated tidal ventilation. A solenoiddriven computer controlled pneumatic ventilator was developed for thispurpose. This device allows for measurements of lung resistance anddynamic elastance during oscillatory ventilation, while monitoring andmaintaining a constant user specified mean airway pressure. Flow (V)into and out of the lung was measured using a pneumotachometer, volume(V) was determined by integration of the flow signal, and transpulmonarypressure (Ptp) was recorded as airway opening pressure referenced toatmospheric pressure.

[0069] The flow pattern chosen for measuring lung function was anoptimal ventilation waveform (OVW) pattern developed by Lutchen et al.(J. Appl. Physiol. 75:478-488, 1993). This pattern represents the sum ofa series of sinusoids selected to provide tidal ventilation whilesimultaneously minimizing signal distortion due to nonlinear effects ofthe respiratory system (Suki et al., J. Appl. Physiol. 79(2):660-671,1995). Lung function was assessed by determining impedance, the ratio ofpressure to flow in the frequency domain, by Fourier analysis. The realand imaginary parts of the impedance signal represent lung resistanceand lung reactance, respectively. Lung resistance is, in turn, equal tothe sum of tissue resistance (R_(ti)) and airway resistance (R_(aw)),while lung reactance is determined by a combination of elastance and gasinertance effects. Thus, in contrast to standard sinusoidal or constantflow ventilation, OVW measurements allow for the determination of airwayresistance, tissue resistance, and dynamic elastance (Edyn) over a rangeof frequencies from a single measurement. This detailed information isuseful for several reasons. Volume reduction is a procedure which hasthe potential for affecting all three of these lung function parameters.In emphysema, volume reduction should reduce R_(aw) by improving airwaytethering, thereby stretching airways open. Because volume reductionincreases tissue stretching, however, it will tend to increase tissueresistance. Total lung resistance, the sum of R_(aw) and R_(ti), cantherefore be variably affected depending upon how LVR individuallyaffects R_(aw) and R_(ti). In most instances, there should be someoptimal range of tissue resection that can produce a substantialdecrease in Raw, but only a small increase in R_(ti). The OVW approachhelps define this optimum. An additional benefit of the OVW approach isthat it provides a non-invasive assessment of lung functionheterogeneity. The presence of heterogeneity, which physiologicallyproduces a positive frequency dependence in lung elastance, can bedetected by the OVW technique (Lutchen et al., J. Appl. Physiol.75:478-488, 1993). In the normal lung, elastance is relatively frequencyindependent since most regions have similar mechanical propertiesleading to uniform gas flow distribution. In a diseased lung, regionaldifferences in impedance to gas flow exist, and elastance increases withincreasing frequency. In emphysema, frequency dependence of elastance isa characteristic finding and reflects regional differences in diseaseseverity. A successful volume reduction targeted at a diseased regionshould reduce heterogeneity and frequency dependence of elastance. Thus,reduction in frequency dependence of elastance can be used as an indexof a successful BLVR procedure, and can be readily determined from theOVW measurement. It is expected that any measurement made immediatelyfollowing BLVR would underestimate the improvement that will becomeevident once a mature scar has formed. At that time, a 25-50%improvement in expiratory flow rates could be observed. Thus, anyfibrin- or fibrinogen-based composition described above is within thescope of the invention if, when applied according to a BLVR procedure,it produces a 25-50% improvement in expiratory flow rates.

[0070] Measurements of lung volumes, quasi-static pressure volumerelationships, and lung resistance and dynamic elastance as functions offrequency were determined in three isolated, naive calf lungs before andafter plication volume reduction. Dynamic recordings were made at 9-10cm H₂O mean transpulmonary distending pressure (PEEP=5 cm H₂O) via theOVW technique at tidal volumes of 10% of measured V_(max). Small leakspresent following plication were sealed with cyanoacrylate glue. Theestimated time between initial and post-reduction recordings was between60 and 90 minutes.

[0071] Pre- and post-volume reduction lung physiology recordings in theisolated calf lung are summarized below in Table 1. TABLE 1 Static andDynamic Lung Mechanics Measured in Isolated Calf Lungs Before andfollowing Plication Lung Volume Reduction Raw (0.2 Hz) Rti (0.2 Hz) EdynVmax (cm H20/L/sec) (cm H20/L/sec) (cm H20/L) (liters) Lung pre post prepost pre post pre post 1 0.42 0.48 1.31 1.30 18.1 22.2 4.4 3.8 2 1.100.85 1.60 2.36 26.3 29.4 3.5 3.1 3 0.82 0.88 3.08 2.92 40.1 36.2 2.9 2.7Mean 0.78 0.74 2.00 2.19 28.2 29.2 3.6 3.2 Std dev 0.34 0.22 0.94 0.8211.1 7.0 0.75 0.56

[0072] These results indicate that, in normal calf lungs, a 10-15%volume reduction (mean 11.1%) produces no significant change in dynamicelastance, airway resistance, or tissue resistance. They furtherdemonstrate that detailed function can be measured in isolated lungsusing the measurement system described herein and that successfulplication volume reduction can be performed on isolated lungs, whichserve as controls for BLVR experiments.

EXAMPLE 2 Fibrinogen-Based Anti-Surfactants

[0073] Mechanical equilibrium across the alveoli and small airways isdetermined by a balance between distending forces, which are exerted bytranspulmonary gas pressure pushing outward, and recoil forces, whichare exerted by parenchymal tissue structures and the surface film liningthe air liquid interface, both of which pull inward and act to promotelung collapse. For the alveoli and small airways to remain patent duringnormal breathing, destabilizing force perturbations must be balanced byintrinsic stabilizing forces. The tendency for the lung to resistdestabilization and atelectasis can be expressed in terms of twobiomechanical properties: the bulk modulus (K) and the shear modulus(p). The value of K is proportional to the lung's ability to resistdistortion resulting from forces directed perpendicular (or normal) to aregion of tissue (Martinez et al., Am. J. Resp. Crit. Care Med.155:1984-1990, 1997), and the value of p is proportional to the lung'sability to resist distortion resulting from shearing forces imposed on aregion of tissue (Stamenovic, Physiol Rev. 70:1117-1134, 1990). Thelarger the values of K and p, the greater the tendency of intrinsicforces within the lung to resist external perturbations and atelectasis.Conversely, any factors which lower K and p tend to promote alveolarinstability and collapse resulting in atelectasis. The values of theshear and bulk moduli depend on both tissue and surface film propertiesand can be quantitatively expressed as (Stamenovic, Physiol Rev.70:1117-1134, 1990):

K=⅓{(B−2)·P _(tis)}+⅓{(3b−1)·P _(γ)}

μ=(0.4+0.1B)·P_(tis)+0.4·P _(γ)

[0074] where B is a normalized elastance for the tissue components(elastin, collagen, and interstitial cells) of the lung; P_(tis) is therecoil pressure of tissue components in the absence of surface filmrecoil; b is a normalized elastance for the surface film at theair-liquid interface; and P_(γ) is the recoil pressure of the surfacefilm in the absence of tissue recoil. In the healthy lung, surfaceforces account for two-thirds to three-quarters of lung recoil, and thusthe contribution of the P_(γ) terms to the bulk and shear moduli areprimarily responsible for determining stability. In emphysema, wheretissue elements are destroyed and exert less recoil, the role of surfaceforces in determining parenchymal stability is of even greaterimportance.

[0075] The primary goal of these experiments was to develop abiocompatible reagent that could be instilled bronchoscopically toproduce site-specific alterations in surface film behavior so as topromote alveolar instability and collapse (i.e., to develop ananti-surfactant). This can be achieved if the liquid film lining thealveoli and small airways undergoes a reduction in bulk and or shearmoduli as a result of chemical modification. In essence, this requires asolution that can alter the surface tension lowering properties ofnative lung surfactant without producing other undesired effects. From abiomedical perspective, the characteristic parameter of the surface filmwhich can be most readily affected by such external chemicalmanipulation is the dimensionless film elastance parameter, b, which isequal to b′·(δγ/δA)(V/A), where γ is the surface tension of the film; Ais the area of the surface over which the film is spread, and b′ is aproportionality constant that varies depending upon the geometry of theregion in question. For normal lung surfactant (δγ/δA)(V/A) (define asb*=b/b′) assumes values of approximately 100-110. As b decreases (i.e.δγ/δA becomes smaller or γ becomes larger) within a given region, thelocal surface forces act more to destabilize rather than stabilize thealveoli. Thus, films with low elastance (relatively flat surface tensionversus surface area profiles) or elevated surface tensions tend topromote destabilization.

[0076] Examples of stable and unstable surface films displaying thesepatterns of behavior are shown in FIGS. 3A and 3B. FIG. 3A shows asurface tension-surface area profile measured by pulsating bubblesurfactometry (PBS) for normal surfactant at 1 mg/ml concentrationisolated from the lung of a normal guinea pig. It demonstrates both apositive film elastance (δγ/δA >0) and low minimum surface tension(γ_(min)<3 dyne/cm). Such a film possesses positive values for thebiophysical parameters b, K, and p and thus would promote stability invivo. In contrast to normal surfactant, a sample isolated from an animalwith acute lung injury and atelectasis following endotoxin infusionshows nearly zero elastance and an elevated minimum surface tension(FIG. 3B). This film has a bulk modulus of less than zero, and thuswould promote alveolar instability and collapse. While these biophysicalfeatures are detrimental in the setting of acute lung injury, theability to impose such features on a film in a controlled fashion isobviously desirable in conducting the present experiments.

[0077] Because the dysfunction observed above occurs in vivo,characterizing the biochemical changes in surfactant that accompany thisdysfunction will suggest biocompatible reagents that are useful in BLVR.Surfactant samples isolated from animals treated with lipopolysaccharide(LPS) are in large part dysfunctional as a result of alterations ininterfacial properties due to mixing with serum proteins, which haveentered the alveolar compartment across the damaged basement membrane(Kennedy et al., Exp. Lung Res. 23:171-189, 1997).

[0078] To determine whether fibrinogen solutions could be added tonative surfactant to produce significant surface film dysfunction, calfsurfactant was isolated by lavage and centrifugation from calf lungsprovided fresh from a local slaughter house. Bovine fibrinogen waspurchased commercially (Sigma Chemical Co., St. Louis, Mo.). Stocksolutions of each reagent were prepared in normal saline, and mixed atmg ratios (fibrinogen to surfactant phospholipid content) of 0.1:1,0.5:1, 1.0:1, 5.0:1, and 10.0:1. The results were compared to mixturesof calf lung surfactant and bovine serum albumin.

[0079] Surface tension recordings were made by pulsating bubblesurfactometry at 37EC, oscillation frequency of 20 cycles/min, and areaamplitude ratio of 72%, continuously, for 5 minutes (Enhoming, J. Appl.Physiol. 43:198-203, 1977). Based on measurements of surface tensionversus surface area, values for the film stability parameter,(γδ/δA)/(A/γ), were determined and expressed as a function of protein tophospholipid concentration ratio. The results are summarized in FIGS. 4Aand 4B. These results suggest that fibrinogen is a more potent inhibitorof surfactant function than albumin and is able to generate markedsurface film instability (expressed in terms of the film stabilitycriteria b*, at protein to lipid concentration ratios of less than 5:1).These concentration ratios are readily achievable in vivo usingconcentrated fibrinogen solutions.

[0080] To test fibrinogen solutions in isolated calf lungs, stocksolutions of bovine fibrinogen (Sigma Chemical, St. Louis, Mo.) in 5 mMTris-HCl (pH 7.4) and partially purified thrombin in 5 mM Tris-HClcontaining 5 mM CaCl₂, were prepared. Mixing studies demonstrated thatratios of fibrinogen to thrombin of between 10:1 to 3:1 (mg:mg) resultedin polymerization within 3-5 minutes. A ratio of 10:1 was selected forwhole lung testing. Isolated calf lungs were cannulated, suspended froma ring clamp, and subjected to baseline lung volume and QSPVCmeasurements as described above. At zero transpulmonary pressure underdirect bronchoscopic visualization, the bronchoscope was wedged in adistal subsegment approximately 5 mm in diameter. The subtended surfacewas then lavaged with 50 mls of fibrinogen solution (10 mg/ml) injectedthrough a P-240 polypropylene catheter passed through the suction port.The fibrinogen solution was stained with several drops of concentratedEvans blue to allow for ready identification of the target region. Thecatheter was then removed, and suction was applied directly through thesuction port of the scope to complete the rinsing procedure. Lavagereturn averaged 28 mls in the 4 lavage procedures performed (56%return). The catheter was then replaced into the affected region and 4mls of thrombin in calcium containing buffer were instilled. A secondlavage and polymerization procedure was then performed in a differentsubsegment. Repeat lung volumes and quasi-static pressure volumeprofiles were then recorded. The results are summarized in Table 2.TABLE 2 Effect of Fibrinogen Instillation and Polymerization on LungVolumes Pre-instillation Volume (L) Post-instillation volume (L) Lung #13.4 2.9 Lung #2 3.1 2.8

[0081] These data indicate that even without the addition of factorXIIIa to promote clot stabilization, reductions in lung volume wereachieved that significantly exceeded the retained volume of polymerizingsolution, indicating that sustained collapse had been achieved.

EXAMPLE 3 Fibrinogen- and Fibrin-Based Solutions Containing GrowthFactors

[0082] Any potential anti-surfactant can be evaluated in the assaydescribed above, which demonstrated that fibrinogen solutions possessmany of the features desired for an anti-surfactant. In addition to thefibrinogen solution described above, one can use solutions that impartadditional characteristics to compositions that can be used to performBLVR in vivo. For example, the fibrinogen solution can be modified tosupport fibroblast growth and to serve as a reservoir for antibiotics.Any modified fibrinogen solution can be used in conjunction with factorsthat promote fibrosis so long as the fibrinogen maintains the ability toinhibit surfactant and undergo polymerization.

[0083] Basic fibroblast growth factor (bFGF) and/or transforming growthfactor-beta (TGFβ) can be added to solutions of fibrinogen, as can anantibiotic mixture of ampicillin and gentamicin, which can be added inamounts sufficient to exceed the minimal inhibitory concentration formost bacteria.

[0084] In vitro studies can be conducted to determine appropriateconcentrations of factor XIIIa relative to fibrinogen and thrombin, andassess how growth factors and antibiotics affect this finalcross-linking step. The surface tension of surfactant fibrinogenmixtures can be measured in vitro using a commercially availablepulsating bubble surfactometer (PBS) unit. Measurements of surfacetension as a function of surface area can be performed at 37EC,oscillation frequency of 20 cycles/min, and a relative surface areachange that approximates that of tidal breathing (δA/A=20-30%).

[0085] Equilibrium and dynamic surface tension recordings can also bemade. Recordings can be performed at 30 seconds, 5 minutes, and 15minutes to ensure that any surface film dysfunction observed initiallyis sustained throughout the measurement period. The stability of eachfilm preparation can be expressed in terms of b*, the dimensionlesssurface film elastance (γ/dA·A/γ) described above normalized to b*values measured for native calf lung surfactant. Mixtures displayingnormalized b* values of <0.2 would be acceptable for additional testingas anti-surfactants.

[0086] Calf lung surfactant can be isolated from whole calf lungs aspreviously described (Kennedy et al., Exp. Lung Res. 23:171-189, 1997),and bovine fibrinogen, bFGF, factor XIIIa transglutaminase, and TGFβ canbe purchased from commercial suppliers (e.g., Sigma Chemical Co., St.Louis, Mo.).

[0087] Surface tension behavior for samples with fibrinogen:surfactantratios ranging between 0.01 to 10 (mg protein:mg lipid) can be preparedin phosphate buffered saline (0.15 M, pH 7.3). The following sixmixtures were chosen because they are representative of mixtures thatmay be useful in vivo, and they contain components of partiallypolymerized fibrin that may exist within the lung during the process ofpolymerization. Thus, they represent the behavior of partiallypolymerized mixtures in vivo and can be assessed to determine whetherthe ability of fibrinogen to inhibit surfactant function changes as itundergoes polymerization.

[0088] Test mixtures will include surfactant (at 1 mg/ml) withappropriate amounts of: (1) fibrin monomer alone; (2) fibrin monomerwith FGF and TGFβ; (3) fibrin monomer with FGF, TGFβ, ampicillin, andgentamicin; (4) fibrinogen with FGF and TGFβ; (5) fibrinogen with FGF,TGFβ, ampicillin, and gentamicin; and (6) fibrinogen with FGF, TGFβ,ampicillin, gentamicin, and thrombin. Recordings will be discontinuedfor the samples that undergo polymerization during surface filmmeasurements (thereby making measurements of γ vs A impossible), and thetime to polymerization will be noted.

[0089] Clot stability can be tested in vitro on solutions ofsurfactant-fibrinogen mixtures containing antibiotics and growth factorswhich demonstrate sustained abnormalities in interfacial properties byPBS measurements. Factor XIIIa can be added to these samples to promoteclot cross-linking. Clot stability at several concentrations of addedfactor XIIIa can be examined by assaying for clot dissolution in 8 Murea (plasma clot lysis time).

[0090] Both fibrin monomers and fibrinogen should cause significantalterations in surface film behavior (normalized b* values<0.2) atprotein:phospholipid ratios>4). Moreover, the addition of thrombin,antibiotics, or growth factors should not markedly alter the ability offibrin compounds to inhibit surfactant function at the concentrationsrequired for these reagents to function in vivo. If these additives domarkedly alter the biophysics of the interaction between surfactant andfibrinogen/fibrin, alternative reagents (or alternative reagentconcentrations), can readily be considered.

[0091] A stable long term state of atelectasis with scarring within thetargeted region is necessary to prevent subsequent partial or completere-expansion following BLVR. This state can be achieved by using abiopolymer that promotes ingrowth of fibroblasts from adjacent regionswithin the lung and causes deposition of extracellular matrix (ECM)components. The procedures described below can be used to examine theability of fibrin polymers containing varying concentrations of growthfactors to stimulate fibroblast ingrowth. More specifically, they can beused to examine the ability of polymers with varying concentrations ofgrowth factors to promote both initial cell attachment and subsequentgrowth.

[0092] Cell culture plates are coated with a mixture of fibrinogen,antibiotics, FGF (both with and without TGFβ), and the mixture ispolymerized by addition of a small amount of thrombin. The plates arethen washed with sterile Eagle's minimal essential medium to removeexcess reagents and thrombin, and sterilized by overnight exposure toultraviolet irradiation. Six types of plates are examined initially: thefirst and second are coated with fibrin polymer, antibiotics, and FGF ateither a low or high concentration; the third and fourth are coated withfibrin polymer, antibiotics, and TGFβ at either low or highconcentration; and the fifth and six are coated in similar fashion butcontain both growth factors at either low or high concentrations.

[0093] Strain IMR-90 (human diploid fibroblasts available from theAmerican Type Culture Collection, Manassas, Va.) are cultured in minimalessential tissue culture medium containing 10% fetal calf serum. Cellsare brought to 80% confluence following initial plating, then harvestedand passed twice in serum free media (MCDB-104, Gibco 82-5006EA, GrandIsland, N.Y.). Established cultures are then sub-cultured onto coated6-well plates at an initial density of 104 cells/ml. Attachmentefficiency (AE) for each coating mixture is assessed at 4 hoursfollowing plating by removing excess media, rinsing the wells in culturefree media, and fixing each well with 70% histologic grade ethanol(Fisher Scientific, Pittsburgh, Pa.). Wells are stained with Geimsa, andthe average number of cells attached per high power field (hpf) isdetermined by light microscopy. Twenty fields per well will be assessedin a blind study. Six wells per coating will be averaged to determinefinal counts, and the results will be compared to those of controlsamples plated on tissue culture plastic. Attachment efficiency will beexpressed as an index (AEI) equal to the ratio of the number cells/hpfin experimental samples to the number of cells/hpf in control samples.Cell growth on each of the six biopolymer mixtures is assessed bydetermining the total number of cells present at 48 hours followingplating. Cell growth is expressed in terms of a growth efficiency index(GEI) equal to the total number of cells at 48 hours for each samplenormalized to the total number of cells at 48 hours of growth on tissueculture plastic. Cell harvesting and counting is performed by removingthe media from each well, and rinsing with calcium/magnesium free Hank'ssolution. The media will be saved for cell re-suspension. One ml of 0.2%trypsin solution is added to each well, and the cells are incubated for2 minutes. Trypsin is then removed, and the adherent cells washed fromthe plate using the previously harvested media, which acts to inhibitfurther trypsin activity. The extent of residual cell adhesion isassessed by direct visualization using an inverted microscope. Residualadherent cells are removed by a second trypsin wash and total cellcounts are obtained using a hemocytometer.

[0094] Cell attachment to a fibrin polymer should be equivalent to, orbetter than, that observed on tissue culture plastic. If cell attachmentis poor using fibrin alone, the fibrinogen will be mixed with 3-5%fibronectin and polymerized. Fibronectin has fibrin binding sites atboth its amino and carboxyl termini, with a central cell binding domainwhich is recognized by most adherent cells expressing P1 integrins.Addition of fibrinogen should result in improved cell adhesion.

[0095] GEI should also be increased in preparations containing bFGF atlow and high concentrations, but may be decreased in preparationscontaining TGFβ because of the suppressant effects of TGFβ on cellproliferation. However, it should be possible to overcome anysuppressant effects observed using TGFβ by using a combination of bFGFand TGFβ. This combination has the potential to promote both cellularingrowth and increase collagen and fibronectin deposition with scarformation. If bFGF is not able to overcome the anti-proliferativeeffects of TGFβ, platelet derived growth factor (PDGF) may be used.

EXAMPLE 4 A Sheep Model for Emphysema

[0096] Work in live animals can help establish the effectiveness,safety, and durability of BLVR. The sheep model of emphysema describedhere displays many of the physiological, histological, and radiographicfeatures of emphysema. In preliminary studies, six adult ewes (weighing27-41 kg) were treated with inhaled nebulized Papain, a commerciallyavailable mixture of elastase and collagenase, administered via amuzzle-mask using two high flow nebulizer systems connected in parallel.The system generates particles 1-5 microns in diameter. Each animalreceived 7,000 units of enzyme in saline over a 90 minute period at 0.3ml/min. Approximately 30-40% of the total dose administered in thisfashion was deposited at the alveolar level. One animal, which receivedsaline according to a similar protocol, served as control.

[0097] All animals underwent detailed measurements of lung functionbefore, and at monthly intervals after, inhalation treatments. Thepost-treatment assessment was continued for 3 months. Recordings weremade following administration of anesthesia during controlledventilation. Transpulmonary pressure was recorded using a pressuretransducer, which recorded the pressure difference between the airwayopening and the intrathoracic pressure measured using an esophagealballoon. Flow at the mouth was measured using a pneumotachographattached to the proximal end of the endotracheal tube. Measurements oflung resistance, static lung compliance, and dynamic lung compliancewere performed. After 3 months, all animals were sacrificed, and lungsections were prepared for histopathological evaluation.

[0098] The results of static and dynamic lung compliance measured priorto exposure to Papain and at 3 months following Papain treatment, aresummarized in FIGS. 5A and 5B. Static lung compliance increasedsignificantly from 0.13 ″ 0.02 to 0.18 ″ 0.03 L/cm H₂O (p=0.012, n=6),indicating disease heterogeneity and gas trapping.

[0099] Physiological changes correlated with a semi-quantitativeassessment of emphysema were also assessed histologically. In a blindstudy, eight sections (one per lobe) from each animal were scored asfollows: 0=no emphysema; 1=mild emphysema; 2=moderate emphysema;3=severe emphysema. A total score was determined as the average fromeight sections prepared from each animal. Total lung resistance tendedto increase with emphysema severity score, although this correlation wasnot statistically significant due to the presence of one outlier, andthe small number of animals studied. Dynamic compliance did correlateinversely with emphysema severity score in a significant fashion (FIGS.6A and 6B).

EXAMPLE 5 In Vivo Application of BLVR

[0100] Induction of emphysema in sheep, as described above, provides anexcellent model in which to test both the safety and efficacy of BLVR.In the studies described below, eight sheep having emphysema wereanalyzed; four did not receive treatment and four were treated withBLVR. Measurements were performed: (1) at baseline prior to papainexposure; (2) eight weeks following papain exposure (at which time allanimals had developed emphysema) and; (3) six weeks following eithersham bronchoscopy without lung volume reduction (control) or BLVRperformed with a fibrinogen-based composition (experimental). Morespecifically, the experimental animals were treated with afibrinogen-based solution containing 5% fibrinogen, which wassubsequently polymerized with 1000 units of thrombin in a 5 mM calciumsolution.

[0101] All animals demonstrated physiological evidence of emphysema withincreased lung resistance, increased dynamic elastance, increased totallung volumes, and changes in static pressure volume relationshipsconsistent with mild to moderate emphysema. Thus, papain therapyadministered via nebulizer, as described above, caused emphysema.

[0102] After six weeks, animals with papain-induced emphysema that didnot receive any therapy had persistent increases in lung resistance(125% at normal breathing frequency compared to pre-treatment baseline)and dynamic elastance (31% at normal breathing frequency compared topre-treatment baseline). Static lung behavior remained markedly abnormalcompared to baseline, with lung volumes increased 33% compared topre-treatment baseline. These results are summarized below in Table 3and shown in FIG. 7. TABLE 3 RL(cm H₂O/L/sec) EL(cm H₂O/L) at RawTreatment at f = 10 b/min f = 10 b/min (cm H₂O/L/sec) Baseline 1.71 ±0.36 10.85 ± 2.78 0.50 ± 0.23 (n = 8) Post-Papain 3.03 ± 0.47 13.51 ±3.81 1.17 ± 0.39 (n = 8) Statistical p = 0.041 p = 0.20 p = 0.029Significance

[0103] In contrast, after six weeks, animals treated with BLVRexperienced a significant reduction in airway resistance, in total lungresistance at normal breathing frequency, in total lung capacity, and inresting lung volumes. These results, which are summarized in Table 4,indicate a significant improvement in lung physiology compared topre-volume reduction, and a significant improvement relative tountreated animals. TABLE 4 RL(cm FRC(liters) TLC (liters) ExperimentalRaw (cm H₂O/L/sec) at resting lung maximum Group H₂O/L/sec) f = 10 b/minvolume lung volume Pre- 0.61 ± 0.31 3.47± 1.14 1.27 ± 0.31 3.31 ± 0.62treatment volume reduction Post- 0.82 ± 0.31 1.85 ± 0.57 0.97 ± 0.212.85 ± 0.71 treatment volume reduction Control 1.14 ± 1.22 3.21 ± 0.970.80 ± 0.31 2.76 ± 0.49 following sham treatment

[0104] Moreover, all animals treated by BLVR tolerated the procedurewell. They were all able to breath without ventilator support within onehour of completion of the procedure, and were eating and drinkingnormally within 24 hours. One of four animals developed a fever, whichlasted two days, and was easily managed with five days of intramuscularantibiotic therapy. No other complications were noted. Thus, thephysiological response to BLVR was very positive.

Other Embodiments

[0105] While the compositions and methods of the present invention areparticularly suitable for use in humans, they can be used generally totreat any mammal (e.g., farm animals such as horses, cows, and pigs anddomesticated animals such as dogs and cats).

[0106] In addition, the compositions described above can be usefullyapplied to a variety of tissues other than the lung. For example, theycan be applied to seal leaks of cerebrospinal fluid; to seal anastomosesof native and prosthetic vascular grafts (including those associatedwith the implantation of prosthetic valves such as mitral valves); indiagnostic or interventional procedures or endoscopic or orthopedicprocedures involving the intentional or accidental puncture of a vesselwall; in plastic surgery; and in highly vascular cut tissue (e.g., thekidneys, liver, and spleen). The compositions described above can alsobe applied to accelerate healing in diabetics and to treat septic woundsof longstanding resistance to standard approaches, includingantibiotic-resistant bacterial infections.

What is claimed is:
 1. A non-surgical method of reducing lung volume ina patient, the method comprising administering, by way of the patient'strachea, to the target region of the patient's lung, an anti-surfactantcomposition, whereafter the target region collapses and one portion ofthe target region adheres to another portion of the target region,thereby reducing the patient's lung volume.
 2. The method of claim 1,wherein the anti-surfactant composition comprises 3-12% fibrinogen. 3.The method of claim 2, wherein the anti-surfactant composition comprisesabout 10% fibrinogen.
 4. The method of claim 2, wherein the fibrinogenis autologous fibrinogen.
 5. The method of claim 2, wherein theanti-surfactant composition further comprises a fibrinogen activator. 6.The method of claim 5, wherein the fibrinogen activator is thrombin, athrombin receptor agonist, or batroxobin.
 7. The method of claim 5,wherein the fibrinogen and fibrinogen activator are administeredseparately.
 8. The method of claim 1, wherein the anti-surfactantcomposition comprises from about 10 mg/ml to about 200 mg/ml fibrin. 9.The method of claim 8, wherein the anti-surfactant composition comprisesfrom about 20 mg/ml to about 200 mg/ml fibrin.
 10. The method of claim9, wherein the anti-surfactant composition comprises from about 20 mg/mlto about 100 mg/ml fibrin.
 11. The method of claim 10, wherein theanti-surfactant composition comprises from about 25 mg/ml to about 50mg/ml fibrin.
 12. The method of claim 8, further comprisingadministering a solution comprising about 3-30 mM CaCl₂.
 13. The methodof claim 1, wherein the anti-surfactant composition further comprises anantibiotic.
 14. The method of claim 1, wherein administering theanti-surfactant composition causes the target region to collapse. 15.The method of claim 1, wherein the method is performed using abronchoscope.
 16. The method of claim 1, wherein the patient is a humanpatient.
 17. The method of claim 1, wherein the patient has emphysema.18. The method of claim 1, wherein the patient has suffered a traumaticinjury to the lung.